Delayed developmental neuro- and immunotoxicity of benzodiazepines.
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Animals↗
Biomedical subjects
M Schlumpf
Publications and source records attributed to M Schlumpf.
Pre- and postnatal ontogeny of [125I]Nle4,D-Phe7-alpha-MSH binding sites in rat brain.
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Aging↗
The effect of prenatal diazepam exposure on TNF-alpha production by rat splenocytes.
Prenatal exposure to diazepam and other benzodiazepines (BDZ) has been found to result in a marked reduction of T-lymphocyte proliferation during postnatal development of rats. In search for pathogenic changes underlying this effect, we investigated the mitogen lipopolysaccharide (LPS) and concanavalin A (ConA) stimulated release of tumour necrosis factor (TNF)-alpha by mixed splenocytes of male offspring from Long Evans rats treated with 1.25 mg/kg per day diazepam from gestational day 14 to 20. In response to LPS, TNF-alpha release was found to be significantly lower in mixed splenocytes of two- and four-week-old treated than in control offspring. However, at eight weeks of age, prenatally diazepam-treated animals showed a significantly higher LPS-induced TNF-alpha release than control rats. Since monocytes/macrophages represent a major source of TNF-alpha, additional experiments were performed on purified spleen macrophages and lymphocytes stimulated with LPS. TNF-alpha release was only detectable in supernatants of adherent spleen macrophages and not in supernatants of lymphocytes. Thus, our data indicate that a disturbance in TNF-alpha release from macrophages is involved in the deficient immune response of prenatally diazepam-exposed rats.
Animals↗
Alterations in interleukin-6 production by LPS- and Con A-stimulated mixed splenocytes, spleen macrophages and lymphocytes in prenatally diazepam-exposed rats.
Prenatal exposure to diazepam leads to a suppression of mitogen or allogen-induced lymphocyte proliferation as well as to a reduced production of tumour necrosis factor (TNF)-alpha from rat splenocytes during postnatal development of rats. We analysed the secretion of interleukin (IL)-6 which occurs at a later stage of the cytokine cascade. Splenocytes of male offspring from Long Evans rats, treated with a daily dose of diazepam (1.25 mg/kg) from gestational day 14 to 20, were stimulated with lipopolysaccharide (LPS) and concanavalin A (Con A). In response to LPS, IL-6 liberation was significantly lower in mixed splenocytes and spleen macrophages of 2 and 8 week old prenatally diazepam-treated rats than in controls. Spleen lymphocyte preparations of prenatally treated animals exhibited a reduction of IL-6 release at 12 h and an increase at 24 h of incubation. At 2 weeks of age, Con A-induced IL-6 production could only be detected in mixed splenocytes; prenatally treated rats were releasing significantly less IL-6 than controls. In 8 week old rats, IL-6 liberation from mixed splenocytes and spleen macrophages was significantly lower in prenatally treated animals than in controls. Spleen lymphocytes presented a complex response picture depending upon incubation conditions. Our data indicate that in prenatally diazepam-exposed rats, the disturbance of cytokine release also extends to cytokines which play an important role in the later phases of immune responses.
Animals↗
Prenatal diazepam induced persisting downregulation of peripheral (omega 3) benzodiazepine receptors on rat splenic macrophages.
Prenatal treatment with a low dose of diazepam (1.25 mg/kg/d, gestational day 14-20) has previously been found to cause longterm impairment of cellular immune responses in rat offspring. A possible site of action of diazepam, i.e., the peripheral-type (omega 3) benzodiazepine receptor, was characterized on splenic macrophages as well as on a rat splenic cell preparation containing mainly lymphocytes. In membranes of both preparations, [3H]PK 11195 bound to a single site which in competition experiments exhibited characteristics of the (omega 3) site. Prenatal exposure to diazepam was followed, at 8 weeks of life, by a marked decrease in maximal binding capacity (Bmax) of spleen macrophage membranes in offspring of both sexes, while membranes of the splenic cell preparation exhibited an increase of the dissociation constant of [3H]PK 11195 at 2 and 8 weeks of life. Both types of delayed effects may reduce the metabolic capacity of these immune cells, in which we have also observed deficits of cytokine release.
Animals↗
Ten-year completed follow-up of percutaneous transluminal coronary angioplasty: the early Zurich experience.
OBJECTIVES: The objective of this communication was to report the long-term follow-up of all the patients treated by Dr. Andreas Gruentzig in Zurich, Switzerland. BACKGROUND: The first patients to undergo percutaneous transluminal coronary angioplasty had the procedure performed by Andreas Gruentzig in Zurich between 1977 and 1980. The method of angioplasty has changed little, and the patients undergoing these first procedures were similar to many patients undergoing angioplasty today; therefore, their long-term outcome is of significant interest. METHODS: All 169 patients with attempted angioplasty during Gruentzig's years in Zurich have now been completely followed up at 10 years. All surviving patients were contacted directly or by telephone interview, and the occurrence of each cardiac procedure and coronary event was analyzed. RESULTS: One hundred thirty-three of the 169 patients underwent successful angioplasty. Ten-year follow-up of this group revealed an overall survival rate of 89.5%. The survival rate was 95% among patients with single-vessel disease and 81% among those with multivessel disease. Patients undergoing unsuccessful dilation and those with failed angioplasty and subsequent bypass surgery had a similar 10-year survival rate. Angiographic restenosis was present in 31% at 6 months. Late restenosis between 6 months and 10 years occurred in eight patients; however, progression of disease in undilated segments occurred in 31 patients. In addition to a better survival rate, patients with single-vessel disease were less likely to have had bypass surgery and were more likely to be angina free at the 10-year follow-up than were patients with multivessel disease (79% vs. 67%). CONCLUSIONS: This earliest angioplasty experience was also the first to demonstrate a difference in outcome between patients with single-vessel and multivessel disease treated with angioplasty. Although angioplasty is now performed in more complex patient subsets, the long-term outcome of these "classic" angioplasty patients should be applicable to similar patients treated today.
Actuarial Analysis↗
Diazepam treatment of pregnant rats differentially affects interleukin-1 and interleukin-2 secretion in their offspring during different phases of postnatal development.
Treatment of pregnant Long Evans rats with benzodiazepines was found to cause alterations in cellular immune responses in their offspring. We now report on changes in interleukin-1 (IL-1) and IL-2 secretion which were analyzed in rats from birth until 12 weeks. Time-pregnant rats were treated with diazepam (1.25 mg/kg/day subcutaneously) from gestational day 14 to 20. Lipopolysaccharide-stimulated release of macrophage-derived IL-1 by spleen cells, determined on D10.G4.1 cells, remained in the control range during the preweaning period (postnatal day 6-28), then decreased in prenatally diazepam-exposed offspring, significantly in males during the postweaning period (postnatal day 34-61) and in both sexes in adults (postnatal day 62-83). Concanavalin A-stimulated release of T lymphocyte-derived IL-2 from spleen cells, determined on CTLL-2 cells, was reduced in male and female offspring during preweaning (postnatal day 3-28) and postweaning (postnatal day 33-55) periods and normalized in adulthood (postnatal day 60-84). The percentage of IL-2 receptor expressing (CD25+) cells was unaffected. From these and our earlier data it is evident that prenatal exposure to low doses of benzodiazepines can result in long-lasting alterations of the cytokine network, as indicated by reduced release of TNF-alpha, IL-1, IL-6, IL-2 and interferon-gamma. The concomitant reduction of peripheral type benzodiazepine receptors on macrophages is discussed as a possible link between prenatal treatment and disturbed function.
Animals↗
In vitro inhibition of cellular immune responses by benzodiazepines and PK 11195. Effects on mitogen- and alloantigen-driven lymphocyte proliferation and on IL-1, IL-2 synthesis and IL-2 receptor expression.
In vitro mitogen-driven lymphocyte proliferation tests (Con A, LPS) on murine lymph node and spleen cells revealed inhibition of T and B cell stimulation by different benzodiazepines and by PK 11195, with IC50 values in the low micromolar range. T cell responses as a consequence of recognition of alloantigens, as measured in mixed lymphocyte cultures (MLC), were affected in an analogous way. In all systems, agonists at peripheral type benzodiazepine receptors (Ro 5-4864 and the non-benzodiazepine compound PK 11195) and diazepam which acts on both, central and peripheral type benzodiazepine receptors, were most potent; clonazepam, a central type agonist, proved about half as active. The central type antagonist Ro 15-1788 failed to antagonize the action of diazepam and clonazepam. Variations among cells from several congenic strains of mice were modest. Cytotoxicity could not be made responsible for drug effects. The most susceptible stage of mitogen-triggered T and B lymphocyte proliferation was found to be at incipience. Radioresistant, adherent spleen cells, upon LPS-stimulation formed only small amounts of the cytokine IL-1. Its release was affected only at very high drug concentrations. Similar small amounts of IL-1 were generated during MLC; in this case, the drugs were about 10 times less potent than in mitogen-induced proliferation assays. Peripheral agonists were more active on IL-1 synthesis. Spleen cells stimulated with Con A and cultivated with the highest concentration of diazepam and clonazepam formed markedly greater amounts of IL-2 than those cultivated in medium, while at this concentration PK 11195 allowed no formation of the lymphokine.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Pre- and postnatal development of high-affinity [3H]nicotine binding sites in rat brain regions: an autoradiographic study.
The ontogeny of high affinity nicotinic cholinergic binding sites was studied in Long-Evans rat brain by in vitro autoradiography, using [3H]nicotine (10 nM) and cold (-)nicotine bitartrate to assess specificity. The first binding sites become detectable in spinal cord and caudal medulla oblongata at gestational day (GD) 12. Until GD 14, labelling spreads throughout lower brainstem, mesencephalon and parts of diencephalon, with higher densities in ventral areas (including the area of developing mesencephalic dopamine neurons). Matrix zones remain unlabelled. Receptor sites appear in the cerebellar anlage by GD 15, and in caudal caudate-putamen by GD 16. During development from late gestational to early postnatal stages, labelling is reduced in many lower brainstem areas and increases in forebrain, in particular in neocortex. Receptor density remains high in thalamus. In neocortex, nicotinic receptor sites are first seen in the subplate layer by GD 20. Labelling of this zone remains prominent until PN 14, when an additional band of increased receptor density is seen in cortical layers III/IV which contain high receptor levels in adulthood. At PN 27, the pattern has become similar to the adult one. The development of [3H]nicotine-binding sites in individual brain regions, with a general caudo-rostral gradient, accompanies cell differentiation and early synapse formation, e.g., in neocortex. The ontogenetic pattern differs in detail from that of muscarinic-cholinergic binding sites. The early presence of binding sites provides a basis for specific actions of nicotine on the fetal brain. As a consequence of the ontogenetic changes, different brain structures become targets for the action of this drug at different stages of development.
Animals↗
Nervous and immune systems as targets for developmental effects of benzodiazepines. A review of recent studies.
Prenatal exposure to benzodiazepines (BDZ) can cause behavioral dysfunctions both in humans and in experimental animals. In addition, prolonged impairment of cellular immune functions is found in rats after low dose BDZ exposure (e.g., diazepam 1.25 mg/kg/day) during part of fetal life [gestational days (GD) 14-20]. Analysis of diazepam and its metabolites in maternal and fetal tissues revealed that in this rat model the drug is no longer present at birth, which excludes direct effects of diazepam during the postnatal period. The main target of BDZ in brain, the GABAA receptor complex, is structurally and functionally heterogeneous. Besides alpha- and beta-subunits, gamma 2- or gamma 3-subunit should be coexpressed for a fully functional BDZ response. Signals of mRNAs encoding for alpha 1, beta 2 and gamma 2 are detected in fetal rat spinal cord and lower brainstem by GD 14 and reach telencephalic regions in later fetal life, reminiscent of BDZ receptor ontogeny. Regional subunit distribution differs from the adult brain, one interesting feature being a preponderance of gamma 2 mRNA throughout fetal life. Since subunit composition influences the sensitivity to BDZ, these data suggest that prenatal effects of BDZ depend upon regional subunit compositions present at different developmental stages. The delayed depression of cellular immune responses in prenatally BDZ-exposed rat offspring during the first 2 postnatal months is accompanied by various changes in immune cell biology. Binding characteristics of the peripheral (omega 3) type BDZ receptor are altered until adulthood (8 weeks). Membranes of spleen cell preparations containing mainly lymphocytes exhibit a decrease of affinity for the peripheral ligand [3H]PK11195, splenic macrophage preparations a decrease of maximal binding capacity. Various defects in cytokine production by macrophages and T lymphocytes were observed: Mitogen-stimulated release of macrophage-derived tumor necrosis factor-alpha (TNF-alpha) and of the T cell-derived interleukin-2 (IL-2) was drastically reduced at 2 and 4 weeks of life and recovered in young adulthood, exhibiting the same time course of depression as lymphocyte proliferation in response to immune stimuli. Interleukin-6 (IL-6) release remained diminished until adulthood. In female offspring, additional alterations were found in splenic noradrenaline turnover after immune stimulation. The mechanisms underlying the breakdown of the cytokine network in prenatally diazepam-exposed offspring, and the long-term consequences are as yet unknown.
Animals↗
Concurrent changes in regional cholinergic parameters and nest odor preference in the early postnatal rat after lead exposure.
The effect of pre- and postnatal lead ingestion on choline acetyltransferase (ChAT) activity, muscarinic cholinergic receptors (mACHR) and on nest odor preference, was investigated in the early postnatal male rat. Long Evans dams were given either 0.15% [LL = low lead] or 0.25% [ML = moderate lead] lead acetate (controls, 0.125% or 0.075% sodium acetate) in their drinking solution from the first day of pregnancy and during lactation. Mean blood lead levels were 55 micrograms/100ml at postnatal day [PN] 6 in ML-treated offspring and 35 micrograms/100ml at PN 9 in LL-treated pups. General health of pups and dams was not affected. Lead-treated offspring showed a reduced preference for or ability to identify the smell of home bedding, when tested at PN 9. A decrease in binding (Bmax) of [3H]N-methyl- scopolamine (NMS) was detected in olfactory bulb and in visual cortex of LL-treated rats at PN 9; the affinity (KD) was unchanged. On the other hand, ChAT-activity of ML-treated offspring was significantly increased in olfactory bulb at PN 6. These results suggest that stage-specific behaviors depending on sensory functions and cholinergic projection systems in related brain areas are sensitive to pre- and postnatal lead exposure.
Animals↗
Prenatal nicotine exposure selectively affects perinatal forebrain aromatase activity and fetal adrenal function in male rats.
Previous studies have revealed effects of prenatal nicotine treatment on fetal plasma testosterone and perinatal sexual brain differentiation in the rat. In an attempt to further elucidate the processes underlying this action of nicotine, we studied the effect of the drug on brain steroid aromatase which converts androgens to estrogens and is known to be important in sexual brain differentiation. Aromatase activity (AA) was measured by the conversion of [1 beta-3H]-androstenedione to estrone in a brain region comprising preoptic, hypothalamic and amygdaloid areas. In untreated animals, the development of AA between gestational day (GD) 18 and postnatal day (PN) 15 was similar in both sexes, except for a significant drop of AA in female brain at PN6, i.e., during the later part of the critical period for sexual brain differentiation. When time-pregnant rats were treated with nicotine delivered by an osmotic minipump for either one week (2 mg/kg/d or 6 mg/kg/d from GD12) or two weeks (6 mg/kg/d from GD8), their male offspring showed a decrease of AA to female levels at PN6, the sex difference existing at this stage thus being abolished. AA of offspring from dams bearing tartaric acid-containing minipumps or sham-operated at GD8 or GD12 was identical to that of untreated controls. No drug effect was seen in female fetuses and offspring. Sex differences in the developmental effect of nicotine may thus involve brain aromatase. An additional sex-dependent effect of nicotine was observed in the male fetal adrenal axis at GD18. Whether the drug effects on the two steroid hormone systems are interrelated, remains to be elucidated.
Adrenal Glands↗
Pre- and postnatal lead exposure affects the serotonergic system in the immature rat brain.
The effect of pre- and postnatal lead exposure on the development of the serotonergic system in striatum and brain stem was investigated. Serotonin and its metabolite 5-HIAA where determined by HPLC-EC. A significant decrease of 5-HT was detected in the brain stem at postnatal day 28. At both days 6 and 28 postnatal, 5-HIAA was reduced in striatum and brain stem. The results provide support to the hypothesis that developing 5-HT neurons are sensitive to relatively low levels of lead exposure.
Animals↗
Regional development of muscarinic cholinergic binding sites in the prenatal rat brain.
The ontogeny of muscarinic cholinergic binding sites was studied in rat fetal central nervous system by in vitro autoradiographic techniques using [3H]N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 microM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic cholinergic sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell populations. High receptor densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.
Animals↗
Prenatal benzodiazepine immunosuppression: possible involvement of peripheral benzodiazepine site.
Treatment of pregnant Long Evans rats with a low dose of diazepam (1.25 mg/kg from gestational day 14-20) produced offspring suffering from suppression of cellular immune responses. Analogous effects were produced by clonazepam, a benzodiazepine (BDZ) with high affinity for the central-type, and Ro 5-4864, a BDZ with selective affinity for the peripheral-type BDZ receptor. Peripheral-type BDZ receptors are found to develop early in fetal life in peripheral organs including primary (thymus) and secondary (spleen) lymphoid organs, in the central nervous system and on immune cells (lymphocytes). In prenatally diazepam-exposed offspring the affinity constant is significantly changed. BDZ and PK 11195 also inhibit mitogen and alloantigen-induced T and B cell proliferation in vitro in adult murine lymphocytes. Diazepam, Ro 5-4864 and PK 11195 were found to be the most active compounds.
Animals↗
Selective retardation of the development of the basal forebrain cholinergic and pontine catecholaminergic nuclei in the brain of trisomy 16 mouse, an animal model of Down's syndrome.
Brain development, examined at embryonic day 17, was retarded in murine trisomy 16 (Ts16). Ts16 is considered to serve as a model of the human trisomy 21 (Down's syndrome) by virtue of the presence in the mouse chromosome 16 of a set of genes located in humans in the segment of chromosome 21 that is requisite to produce the phenotypic features of Down's syndrome when present in triplicate. In addition to a reduction in brain size and cortical thickness, we observed a severe reduction throughout the brain in the density of muscarinic receptors, assessed by autoradiographic detection of specifically bound tritiated N-methyl-scopolamine, and by the failure of the development of the differentiated pattern of receptor distribution in the brainstem. The effect of gene dosage was also examined on specific neuronal populations. The distribution of acetylcholine esterase (AChE)-, tyrosine hydroxylase (TH)- and 5-hydroxytryptamine (5-HT)-positive cells in the trisomic brain was similar to that observed in chromosomally balanced littermates. On the other hand, the number of AChE-positive cells was 60-70% of the estimates in littermate controls in regions containing the septum, the vertical and horizontal limbs of the diagonal band and the basal forebrain cholinergic nuclei. Similarly, the number of TH-positive cells was reduced by about 30% in the pons. In contrast, in the trisomic foetuses the number of TH-positive cells in the mesencephalon and the diencephalon was similar to that in littermate controls, while that of 5-HT-positive cells in the mesencephalic nuclei was only slightly affected, if at all. Ts16 results, therefore, in a selective retardation of some neuronal systems, and this may lead to a perturbation of brain development. Furthermore, the systems whose development was retarded selectively are those which in Down's syndrome adults exhibit pronounced deficits of cells that--in case the murine Ts16 is a valid model--may also involve developmental disorders.
Acetylcholinesterase↗
Prenatal diazepam induced persisting depression of cellular immune responses.
Treatment of pregnant Long Evans rats with a low dose of diazepam (1.25 mg/kg per day s.c.) from gestational day (GD) 14 to 20 resulted in severe and long lasting depression of cellular immune responses in male and female offspring. T lymphocyte proliferation, induced by allogeneic stimulation in mixed lymphocyte culture (MLC) or geneic stimulation in mixed lymphocyte culture (MLC) or mitogenic stimulation (concanavalin A), decreased by 50 % or more over a postnatal period of about 2 months. Treatment of the pregnant dam during the early fetal period, from GD 12 to GD 16, did not significantly affect these immune parameters, whereas treatment during later gestation, from GD 16 to 20, significantly affected T lymphocyte function. Clonazepam, a benzodiazepine with high affinity for the central type benzodiazepine site, also affected cellular immune response in offspring. Our data indicate that benzodiazepine treatment during the fetal period may result in persistent postnatal deficiency of cellular immune responses. The relative role of central and peripheral type benzodiazepine receptor and possible interactions with maternal and fetal pituitary - adrenocortical systems are discussed.
Animals↗
Diazepam effects on the fetus.
Treatment of time-pregnant Long Evans rats with 1.25 mg/kg s.c. diazepam (2.5 mg/kg in Sprague Dawley rats) from gestational day 14 to 20 produced transient depression of an olfactory guided behavior (nest odor behavior) in suckling offspring. Enhanced drug sensitivity to diazepam was seen in adult male and female off-spring as indicated by increased temperature depression. In addition, increased sensitivity to an opiate (morphine) was noted for the female offspring in the tail flick test. Treatment of the pregnant dam with diazepam or clonazepam, a benzodiazepine with selective affinity for the central benzodiazepine receptor, resulted in a marked depression of cellular immune responses in the offspring of both sexes up to 2 months of age. Drug treatment during early fetal period (GD 12-16), at a time central benzodiazepine receptors are not present in all brain regions of the fetal brain, did not affect the quality of cellular immune responses, whereas treatment from GD 16 to 20 was effective. Prenatal diazepam effects are discussed in view of presence and functionality of both central and peripheral benzodiazepine binding sites in the fetus.
Animals↗